HISTOCHEMISTRY: RATIONALE, PROCESSING, STAINING - BIOLOGY - 2025

The histochemical is a useful tool in the study of the morphology of various biological tissues (plants and animals) due to its reaction principle of tissue components such as carbohydrates, lipids and proteins, among others, chemical dyes.

This valuable tool allows not only to identify the composition and structure of tissues and cells, but also the various reactions that occur in them. Likewise, possible tissue damage caused by the presence of microorganisms or other pathologies can be evidenced.

Histochemical stains. Nile virus, Gram positive and Gram negative bacteria (Gram), Histoplasma capsulatum (Grocott), Mycobacterium tuberculosis (Ziehl Neelsen). Source: Pixinio.com/Wikipedia.org/Nephron / CDC / Dr. George P. Kubica

Histochemistry, from past centuries has provided important contributions, such as the demonstration of the existence of the blood-brain barrier by Paul Ehrlich. This was possible because the brain of the experimental animal used by Ehrlich was not stained with aniline, which is a basic dye.

This led to the use of various dyes such as methylene blue and indophenol, in order to stain the different types of cells. This finding led to the classification of cells into acidophilic, basophilic and neutrophilic, according to their specific staining.

Recent studies have applied this technique to show the presence of various compounds, including phenols, as well as carbohydrates and non-structural lipids, in the tissues of the Litsea glaucescens species, better known as laurel. Finding these, both in the leaf and in the wood.

Likewise, Colares et al, 2016, identified the plant of medicinal interest Tarenaya hassleriana, using histochemical techniques. In this species the presence of starch, myrosine, as well as phenolic and lipophilic compounds was evidenced.

Basis

Histochemistry is based on the staining of cellular structures or molecules present in the tissues, thanks to their affinity with specific dyes. The reaction of coloration of these structures or molecules in their original format, is later visualized in the optical microscope or electron microscope.

The specificity of the staining is due to the presence of ion-accepting groups present in cells or tissue molecules.

Finally, the objective of histochemical reactions is to be able to show it through staining. From the largest biological structures to the smallest of tissues and cells. This can be achieved thanks to the fact that the dyes react chemically with the molecules of the tissues, cells or organelles.

Prosecution

The histochemical reaction could involve steps prior to performing the technique, such as fixation, embedding and cutting of the tissue. Therefore, it must be taken into account that in these steps the structure to be identified can be damaged, yielding false negative results, even if it is present.

Despite this, the prior fixation of the tissue carried out properly is important, since it prevents autolysis or cell destruction. For this, chemical reactions are used with organic solvents such as: formaldehyde or glutaraldehyde, among others.

The inclusion of the fabric is done so that it maintains its firmness when cut and thus prevents it from deforming. Finally, the cut is made with a microtome for the study of samples by optical microscopy.

Additionally, before proceeding with the histochemical staining, it is recommended to incorporate external or internal positive controls in each batch of tests. As well as the use of specific dyes for the structures to be studied.

Histochemical stains

From the emergence of histochemical techniques to the present, a wide range of stains have been used, including those most frequently used such as: Periodic acid Schiff (PAS), Grocott, Ziehl-Neelsen and Gram.

Likewise, other dyes have been used less frequently such as India ink, orcein or Masson's trichrome stain, among others.

Periodic Acid Schiff (PAS)

With this coloration, molecules with high carbohydrate content can be observed, such as: glycogen and mucin. However, it is also useful for the identification of microorganisms such as fungi and parasites. In addition to certain structures (basement membrane) in the skin and other tissues.

The basis for this staining is that the dye oxidizes the carbon bonds between two nearby hydroxyl groups. This produces the release of the aldehyde group, and this is detected by the Schiff's reagent, giving off a purple color.

The Schiff reagent is composed of basic fuchsin, sodium metabisulfite and hydrochloric acid, these components being responsible for the purple coloration, when aldehyde groups are present. Otherwise a colorless acid is generated.

The intensity of the coloration will depend on the amount of hydroxyl groups present in the monosaccharides. For example, in fungi, basement membranes, mucins and glycogen the color can go from red to purple, while the nuclei stain blue.

Grocott

It is one of the stains with the highest sensitivity in the identification of fungi in paraffin-embedded tissues. This allows the identification of the various fungal structures: hyphae, spores, endospores, among others. Therefore, it is considered a routine stain for the diagnosis of mycosis.

It is especially used in the diagnosis of pulmonary mycoses such as pneumocystosis and aspergillosis caused by some fungi of the genera Pneumocystis and Aspergillus, respectively.

This solution contains silver nitrate and chromic acid, the latter being a fixative and colorant. The rationale is that this acid produces the oxidation of hydroxyl groups to aldehydes by mucopolyacharides present in fungal structures, for example in the cell wall of fungi.

Finally, the silver present in the solution is oxidized by the aldehydes, causing a black coloration, which is called the argentafin reaction. Contrast dyes such as light green can also be used and thus the fungal structures will be observed in black with a light green background.

Ziehl-Neelsen

This staining is based on the presence of acid-alcohol resistance, partially or totally, in some microorganisms such as the Nocardia, Legionella and Mycobacterium genera.

The use of this stain is recommended, because the cell wall of the previously mentioned microorganisms contains complex lipids that hinder the penetration of the dyes. Especially in samples from the respiratory tract.

In it, strong dyes such as carbol fuchsin (basic dye) are used and heat is applied so that the microorganism can retain the dye and does not discolor with acids and alcohols. Finally, a methylene blue solution is applied to color the structures that have become discolored.

The presence of acid-alcohol resistance is observed in structures stained red, while structures that do not resist fading are stained blue.

Gram and chinese ink

The Gram is a very useful stain in the diagnosis of bacterial and fungal infections, among others. This staining makes it possible to differentiate between Gram-positive and Gram-negative microorganisms, clearly showing the differences that exist in the composition of the cell wall.

While India ink is a stain that is used to contrast structures that contain polysaccharides (capsule). This is because a ring is formed in the environment, being possible in Cryptococcus neoformans.

Orcein

With this staining, the elastic fibers and chromosomes of various cells are colored, allowing the evaluation of the maturation process of the latter. For this reason, it has been very useful in cytogenetic studies.

This is based on the uptake of the dye by the negative charge of molecules such as DNA, present in the nuclei of a wide variety of cells. So these are stained blue to dark purple.

Masson's trichrome

This stain is used to identify some microorganisms or materials that contain melanic pigments. This is the case of mycoses, caused by dematiaceous fungi, pheohifomycosis and in black grain eumycetoma.

Final thoughts

In recent years there have been many advances in the creation of new diagnostic techniques, where histochemistry is involved but linked to other fundamentals or principles. These techniques have a different purpose, as in the case of immunohistochemistry or enzymohistochemistry.

References

1. Acuña U, Elguero J. Histoquímica. An. Chem. 2012; 108 (2): 114-118. Available at: are.iqm.csic.es
2. Mestanza R. Frequency of PAS, Grocott and Ziehl-Neelsen histochemical stains used for the identification of microorganisms, performed in the Pathological Anatomy Service of the Eugenio Espejo Specialty Hospital in 2015.. Central University of Ecuador, Quito; 2016.Available at: dspace.uce.edu
3. Tapia-Torres N, de la Paz-Pérez-Olvera C, Román-Guerrero A, Quintanar-Isaías A, García-Márquez E, Cruz-Sosa F. Histochemistry, total phenol content and antioxidant activity of litsea leaf and wood glaucescens Kunth (Lauraceae). Wood and Forests. 2014; 20 (3): 125-137. Available at: redalyc.org
4. Colares, MN, Martínez-Alonso, S, Arambarri, AM. Anatomy and histochemistry of Tarenaya hassleriana (Cleomaceae), a species of medicinal interest. Latin American and Caribbean Bulletin of Medicinal and Aromatic Plants 2016; 15 (3): 182-191. Available at: redalyc.org
5. Bonifaz A. Basic medical mycology. 4th edition. Mexico: McGraw-Hill Interamericana editores, SA de CV 2012.
6. Silva Diego Filipe Bezerra, Santos Hellen Bandeira de Pontes, León Jorge Esquiche, Gomes Daliana Queiroga de Castro, Alves Pollianna Muniz, Nonaka Cassiano Francisco Weege. Clinico pathological and immunohistochemical analysis of spindle cell squamous cell carcinoma of the tongue: a rare case. Einstein (São Paulo) 2019; 17 (1): eRC4610. Available from: scielo.br